Summary: The purpose of the research is to study the cellular and molecular mechanisms of adaptation to stress with emphasis on the regulation of the various components of the hypothalamic pituitary adrenal (HPA) axis. This includes the expression of hypothalamic corticotropin releasing hormone (CRH) and vasopressin (VP), pituitary CRH and V1b VP receptors, and adrenal steroidogenesis. The research employs a combination of in vivo and in vitro such as measurement of gene expression, receptor binding, signaling pathways in brains, adrenals and pituitaries of rodents subjected to experimental stress models or genetic modifications, and studies in cell lines. In vivo studies on the regulation of CRH expression have shown that the increases in CRH transcription during prolonged stress are transient. This decline is independent of the increases in circulating glucocorticoids but it is associated with increased expression of inducible cAMP early repressor (ICER), a repressor isoform of cAMP responsive element modulator (CREM), in CRH cells of the PVN. Extension of these studies demonstrated time dependent increases in phospho-CREB (30 min) and ICER (3h) in the PVN following stress or in the hypothalamic cell line, 4B, after incubation with forskolin. Induction of ICER at 3 h was associated with CREM recruitment by the CRH promoter and a reduction in Poll II recruitment. Co-transfection of ICER in showed that repressor markedly inhibits CRH promoter activity. These data supports a role of an intracellular feedback mechanism mediated by the induction of ICER in the limitation of CRH transcription during stress. Studies were performed to examine the role of the reproduction-related hormones oxytocin and prolactin on the adaptation of the HPA axis to stress during late pregnancy and lactation. Preliminary experiments in this laboratory suggested that the postulated inhibitory effect of oxytocin on HPA axis activity depends on the prevailing levels of estrogen. To elucidate this question, the effects of intracerebroventricular (icv) oxytocin on HPA axis responses to 1 h restraint stress were studied in ovariectomized rats receiving slow release implants yielding low, intermediate and high plasma estradiol levels. Oxytocin infusion had no effect on basal plasma corticosterone or ACTH but it inhibited responses to stress depending on presence of circulating estradiol. Consistent with the plasma hormone responses, in situ hybridization studies showed that central oxytocin had no effect on basal or stress stimulated CRH hnRNA and CRH mRNA in the PVN, or POMC mRNA in the pituitary in rats receiving low doses of estradiol, but significantly decreased levels in the presence of high estradiol. The study shows that the ability of central oxytocin to inhibit HPA axis activity depends on the levels of circulating estradiol. Studies in progress examining the mechanism by which prolactin can modulate HPA axis activity show that icv injection of prolactin induces phosphorylation of ERK in the hypothalamic PVN. Hypothalamic cell lines, which express CRH, were shown to have prolactin receptors. In these cells prolactin induced ERK phosphorylation and small increases in CRH promoter activity. These findings may be relevant to the pathogenesis of psychiatric disorders associated with reproduction and reduced HPA axis activity such as post-partum depression and premenstrual syndrome. Studies on the VP and CRH receptors have focus on the interaction between these receptors. Regulation of ACTH secretion involves strong interaction between activation of V1b receptors and corticotropin releasing hormone receptor type 1 (CRHR1), but whether there is physical interaction between these receptors is currently unknown. This question was addressed using bioluminescence resonance energy transfer (BRET) and immunoprecipitation techniques to study dimerization in living cells. Energy transfer from V1bRrluc to either, V1bYFP, CRHR1YFP or the unrelated bradykinin 2 receptor-YFP (B2RYFP) was very low and levels were unchanged by cotransfection with wild type V1bR or following incubation of the cells with the ligands, CRH or VP suggesting no physical interaction between these receptors. In contrast to the BRET data, co-immunoprecipitation using receptors tagged with c-myc and Flag epitopes demonstrated specific homodimerization of the V1b receptor and heterodimerization of the V1b receptor with both the OTR and CRHR1 receptors, suggesting that the position of the rluc and YFP tags impaired the BRET signal. Studies in course focus on the functional significance V1b and CRHR1 receptor dimerization. Vasopressin produced by parvocellular neurons of the PVN potentiates the stimulatory effect of CRH on pituitary ACTH secretion acting through plasma membrane receptors of the V1b subtype (V1bR). The expression of parvocellular VP as well as that of pituitary V1bR increases during chronic stimulation of the hypothalamic pituitary adrenal axis, suggesting that VP plays a critical role on long term adaptation to stress. Recent studies using VP antagonists and VP and V1bR deficient animal models have shown little impact of vasopressinergic blockade on HPA axis activity during chronic stress, suggesting that VP has additional roles, such as controlling the number of pituitary corticotrophs. This possibility was studied using prolonged adrenalectomy, which induces increase in vasopressinergic activity and corticotroph mitogenesis, to examine the effect of VP receptor blockade by minipump infusion of the peptide antagonist, dGly[Phaa1,D-tyr(et),Lys,Arg]VP, on the number of cells incorporating bromodeoxyuridine (BrdU) and cells stained for ACTH in the anterior pituitary. Adrenalectomy increased the number of BrdU labeled cells by about 3-fold compared with controls, and infusion of the V1 antagonist completely prevented the effect of ADX. The number of ACTH stained cells also increased after 4 week ADX, but in contrast to BrdU incorporation, the increase was not affected by the V1 antagonist. Unexpectedly, there was only minor co-localization of BrdU uptake in ACTH positive cells and this was unaffected by ADX or V1 antagonist infusion. Incubation of pituitary cells primary cultures with VP for 48h increased the number of cells incorporating BrdU by about 30%. These studies show that VP has multiple roles in pituitary function, including upregulation of the V1b receptor, potentiation of CRH-stimulated ACTH secretion and trophic actions. These data suggest that while in acute conditions VP facilitates ACTH secretion, in chronic situations, VP contributes to mitogenic activity in the pituitary without increasing the number of ACTH containing corticotrophs. The lack of colocalization of ACTH in mitotic cells, suggest that recruitment of corticotrophs during adrenalectomy occurs from undifferentiated cells.